Synthesis, molecular docking, and in-vitro studies of pyrimidine-2-thione derivatives as antineoplastic agents via potential RAS/PI3K/Akt/JNK inhibition in breast carcinoma cells

In the present investigation, derivatives from (2–6) containing pyrimidine-2-thione moiety incorporated with different heterocycles such as pyrazoline, phenyl pyrazoline, and pyrimidine were synthesized using different methods. These pyrimidine-2-thione derivatives were evaluated in-silico for their capability to inhibit the H-RAS-GTP active form protein with insight to their pharmacokinetics properties. According to our findings, compound 5a was selected for in vitro studies as it has the in-silico top-ranked binding energy. Furthermore, compound 5a induced apoptosis to panels of cancer cell lines with the best IC50 on MCF-7 breast cancer cells (2.617 ± 1.6 µM). This effect was associated with the inhibition of phosphorylated RAS, JNK proteins, and PI3K/Akt genes expression. Thus, compound 5a has upregulated p21 gene and p53 protein levels. Moreover, 5a arrested the cell cycle progression at the sub-G0/G1 phase. In conclusion, the synthesized compound, 5a exhibited potent antineoplastic activity against breast cancer cell growth by targeting RAS/ PI3K/Akt/ JNK signaling cascades.

Method A: Conventional heating. General method for the synthesis of compounds 1a, b. Compounds 1a, b were synthesized as described by Kappe 25 .
General method for the synthesis of chalcones (2a, b). A mixture of compound 1 (25 mmol), vanillin (3.80 g, 25 mmol), and sodium hydroxide (2 g, 50 mmol) in ethanol (50 mL) were stirred at room temperature for 24 h (TLC control). Then, the reaction mixture was poured into ice water, filtered off, and dried to afford 2a, b 26 .
ADMET in-silico prediction. The Swiss Institute of Bioinformatics' online tool SwissADME (http:// www. swiss adme. ch/) was used to examine the pharmacokinetics and drug-likeness prediction of the newly synthesized pyrimidine-2-thione derivatives. SwissADME's SMILES generator was used to transform the compound's 2D structural model into SMILES, which was then analyzed to determine the compound's different ADMET qualities in terms of pharmacokinetics and drug-likeness 31 .
Biology section. Measurement of cell viability by MTT. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure cell viability according to a prior study 32 . The cells were grown in 96-well plates at a density of 1 × 10 4 cells/well, treated for 48 h with varied 5a (0-200 µM) or the reference drug DOX (0-100 µM) dosages, and then exposed to analysis. The culture medium was taken out after 48 h of incubation, and the cells were given two gentle washes with 1× ice-cold phosphate-buffered saline (PBS). The MTT (10×) has been diluted to a 1× solution in the culture medium (1 volume 10× MTT in 9 volumes medium) and 200 μl was added to each well. The microplate has been kept in a cell culture incubator at 37 °C for 4 h. Afterthat, 180 μl of the MTT solution was taken out and 100 μl of DMSO was put to each well. The microplate was incubated for 15 min with shaking at room temperature. The absorbance of each well was ultimately measured at 630 nm using a Model 680 microplate reader (Bio-Rad, CA, USA) 33 .
Cell morphology. In a six-well plate, 1 × 10 5 MCF-7 cells were seeded and incubated for 24 h then treated with the IC 50 dose of the selected compound. Using an inverted light microscope (Olympus, USA), the morphological alterations of the treated and untreated cells were evaluated and documented after 48 h of incubation 34 .
Cell cycle analysis by flow cytometry. Using flow cytometry, cell cycle analysis was carried out via a documented method of 35 with a few changes. After trypsinization, the MCF-7 cells were centrifuged at 4500 rpm for 5 min, followed by two washings, resuspension in 1× PBS, fixation with ice-cold absolute ethanol, and incubation at 20 °C for 24 h. Following two PBS washes, the cells were then resuspended in (0.05 mg/mL) propidium iodide (PI) 32 solution containing 50 µL (0.2 mg/mL) of RNase A, and 0.1% v/v Triton X-100 in PBS. Then, the cells were incubated at room temperature for 30-60 min in the dark. Finally, the pellet was washed with 1⤫ PBS and resuspended in 300 µL 1× PBS, and subjected to analysis on Accuri C6 flow cytometer (Becton Dickinson, Franklin Lake, BD, USA) equipped with PE X FL2 channels 33 . www.nature.com/scientificreports/ RT-PCR analysis. The total RNA was isolated from the MCF-7 cells using Trizol (Cat# R2072, ZYMO RESEARCH CORP. USA) according to the manufacturer's instructions. Its quantity and quality were assessed using a Beckman dual spectrophotometer 28 . The cDNA was performed using a miScript Reverse Transcription Kit (QIAGEN) Kit (218061) and a miScript SYBR Green PCR Kit was used for mRNA detection with quantification (218073). Reverse transcription was running on a StepOne system (Applied Biosystems, Foster City, CA, USA) in a total volume of 10 μL containing 5 μL of the miScript RT enzyme and 10 μL of the total RNA. Real-time PCR for mRNA was performed on the same system in a total volume of 20 μL containing 10 μL of a 2× QuantiTect ® SYBR Green PCR Master Mix and 10 ng of the cDNA template. The PCR thermal cycling conditions for the mRNA were 2 min at 50 °C, 30 s at 95 °C, 40 cycles of 95 °C for 5 s, and 60 °C for 34 s. The expression of the mRNA target genes was normalized to the expression of human GAPDH quantified using the 2 −ΔΔCt method 36 . Table 1 contains a list of the primers used in the research.
Immunoblotting analysis. Western blot analysis was conducted according to 37  The chemiluminescent substrate (Clarity TM Western ECL substrate Bio-Rad cat#170-5060) was applied to the blot according to the manufacturer's recommendation and the chemiluminescent signals were captured using a charge-coupled device (CCD) camera-based imager. Image analysis software was utilized to compare the target protein's band intensity to those of the control sample β-actin by protein normalization on the (ChemiDoc MP imager (https:// www. bio-rad. com/ en-us/ produ ct/ chemi doc-mp-imagi ngsys tem? ID= NINJ8 ZE8Z).
Statistical analysis. Data have been analyzed using the GraphPad Prism software (San Diego, CA, USA) (Graph-Pad Prism 6, https:// www. graph pad. com/ scien tific-softw are/ prism/). The experimental data are expressed as mean ± SE. The significance of the difference between the treated and the control groups were analyzed using the t-test to compare these groups. The acceptable significance was recorded when the p-value was < 0.05.
The condensation of chalcones 2a, b with urea in boiling ethanolic hydrochloric acid afforded 3a, b (Fig. 2). The IR spectrum of these compounds showed significant absorption bands at 1568-1590 cm −1 characteristic of C=N group, absorption band in the region within the range of ν 1605-1764 cm −1 corresponding to (C=O) group and absorption bands at 3640-3641 cm −1 characteristic of the OH group. The 1 H NMR spectrum of compound  (Fig. 2). The 1 H NMR spectrum of compound 4a exhibited singlet signal at δ 5.04 ppm due to CH of the new pyrimidine ring and singlet signals at δ 9.73 ppm attributed to NH proton in the new pyrimidine ring. Its 13 C NMR spectrum revealed novel three signals at δ 163.93, 174.73 and 181.09 respectively attributed to C-NH, C=N and C=S of new pyrimidine ring. The 1 H NMR spectrum of compound 4b exhibited singlet signal at δ 5.26 ppm due to CH proton of the new pyrimidine ring and singlet signals at δ 9.38 ppm for NH proton. Its 13 C NMR spectrum exhibited three signals attributed to C-NH, C=N and C=S groups in pyrimidine ring at δ 165.23, 175.02 and 180.74 respectively.
The cyclo-condensation of chalcones 2a, b with phenyl hydrazine in acetic acid led to the formation of the phenyl pyrazoline derivative 5a, b (Fig. 3). The 1 H NMR spectra of compound 5a offered a new doublet signal at δ 1.90 ppm corresponding to CH 2 pyrazoline and a new triplet signal at δ 3.80 attributed to CH pyrazoline. Its 13 C NMR spectrum exhibited three new signals at δ 32.18, 58.56 and 147.72 attributed to CH, CH 2 and C=N of pyrazoline respectively. The 1 H NMR spectra of compound 5b showed doublet signal at δ 1.86 ppm attributed to CH 2 pyrazoline and triplet signal at δ 3.80 due to CH pyrazoline. Its 13 C NMR spectrum showed three new signals at δ 36.61, 54.70 and 149. 64 due to CH, CH 2 and C=N respectively.
Further modification of compounds 2a, b with hydrazine hydrate led to the formation of pyrazoline derivative 6a, b (Fig. 3). The 1 H NMR spectrum of compound 6a exhibited a new doublet signal at δ 2.19 ppm corresponding to the CH 2 pyrazoline, a new triplet signal at δ 5.64 attributed to CH pyrazoline and new singlet signals at δ 10.28 ppm ascribable to NH pyrazoline. Its 13 C NMR spectrum revealed new three signals at δ 43.33, 50.91 and 146.14 attributed to CH 2 , CH and C=N of pyrazoline ring respectively. Moreover, the 1 H NMR spectrum of compound 6b showed doublet signal at δ 2.10 ppm for CH 2 pyrazoline, triplet signal at δ 5.59 attributed to CH pyrazoline and singlet signals at δ 10.08 ppm ascribable to NH pyrazoline. Its 13 C NMR spectrum showed three new signals at δ 42.04, 50.63 and 145.85 due to CH 2 , CH and C=N of pyrazoline ring respectively.
Ultrasonic irradiation has been determined as the most eco-friendly technique that can be used to accelerate the reaction rate and ensure superior yield during organic synthesis 38 . Therefore, a series of compounds 3-6 were synthesized via ultrasonic irradiation and afforded the same synthesized compounds obtained from the conventional method (melting point, mixed melting point, TLC, and spectral analyses) at higher yields in a short time (Table 2). study Molecular docking study (in-silico). One of the main signaling pathways linked to tumor development is RAS/JNK and RAS/PI3K/Akt. As a result, molecular docking was used in the current study to investigate the potential H-RAS novel drug candidates. The docking scores of newly synthesized compounds against H-RAS, GTP active form target protein ranged from -5.6 to − 11.16 kcal/mol, respectively. According to the results of

Studies on ADME/T and the Lipinski rule.
To certify a medicine and its efficacy as a top candidate against any disease, ADME/T is necessary. The partition coefficient (cLogP), donor hydrogen-bond, and drug similarity were all calculated using in-silico Physio-chemical methods, and they were all assessed using Lipinski's rule of five 39 . Also, studies on bioavailability and pharmacokinetics have been carried out to perform such clinical trials on these novel synthesized pyrimidine-2-thione derivatives. The topological polar surface (TPSA) must be less than < 140 Å 2 for great oral bioavailability 40    www.nature.com/scientificreports/ A drug candidate must first pass a toxicity risk assessment to be considered for drug development. Low toxicity and side effects are indicative of a medicine's high therapeutic index. Due to this, an in-silico toxicity analysis was carried out, with the outcomes displayed in Table 4. Surprisingly, none of the substances proved carcinogenic.
Additionally, an AMES toxicity analysis was conducted, and all compounds (3a-6b) tested negative, indicating that they have no mutagenic effects. Computed LD 50 levels in the oral rat acute toxicity models have been calculated for further investigation of the in-vivo anticancer activities, and the compounds appear to be quite safe (2.42-2.76 mol/kg) 41 .
According to the previous results, the best-docked molecule 5a that impact a great inhibition to H-RAS, GTP active form target protein also, showed perfect in-silico physio-chemical, pharmacokinetic and bioavailability without any toxicity and carcinogenicity could be a potential new anticancer drug candidate.

In vitro studies. Growth inhibition of different human cancer cell lines by compound 5a.
In the present study, the cytotoxic effect of the synthesized compound 5a, and the chemotherapeutic drug, DOX was revealed by MTT assay on different human cancer cell lines including breast (MCF-7 and MDA-MB-231), colorectal (Caco-2), and pancreatic (PANC-1) cancer cells as well as normal cells (WISH). Compound 5a caused dose-dependent suppression of MCF-7, MDA-MB-231, Caco-2, and PANC-1 cancer cell proliferation with IC 50 values of 2.617 ± 1.6, 6.778 ± 2.2, 14.8 ± 2.5 and 23.58 ± 2.4 µM respectively (Fig. 5A). According to our finding, compound 5a exhibited the lowest IC 50 value against MCF-7 cells indicating that they were more sensitive to this compound than other malignant cell lines compared with the cytotoxic effect of DOX that exhibited an IC 50 value equal to 2.261 ± 1.4 µM (Fig. 5B). On the other hand, compound 5a exhibited less cytotoxicity to the normal cell line with an IC 50 of 180.5 ± 2.7 µM in comparison with cancer cell lines, which proved its safety on normal cells. Contrarily, DOX showed a significant toxic effect against normal cells with an IC 50 of 24.49 ± 2.1 µM. Therefore, we extended our study using the MCF-7 cells.

Alternation of the morphological features of MCF-7 cells by compound 5a.
In this study, MCF-7 cells treated with an IC 50 of 5a showed detectable morphological signs of apoptosis such as cell rounding and shrinkage with decreased cell number detachment, and cytoplasmic condensation. However, the untreated cell morphology appeared normal and confluent. These findings provided more information regarding the capacity of compound 5a to trigger cell death (Fig. 6).

Induction of cell cycle arrest of the MCF-7 cells by compound 5a.
Cell cycle analysis was conducted on breast cancer cells to better understand the mechanism of the 5a-mediated tumor growth inhibition, and the findings are shown in Fig. 7. In the MCF-7 untreated cells, oncogenic RAS-induced proliferative signals resulted in the upregulation of transcriptional factors required for cell cycle entrance and cellular progression. Additionally, the metabolic stability of cyclin D1 was affected by the suppression of glycogen synthase kinase (GSK) 3β, which is correlated with continuous ubiquitination and proteasomal degradation of cyclin D1 via PI3K-dependent phosphorylation 17 . Moreover, oncogenic RAS accelerates cell cycle progression by reducing the inhibition caused by antigrowth signaling pathways which inhibit cyclin-dependent kinase (CDK) inhibitors like p21 42 . Oncogenic RAS may contribute to gene instability in cancer via the promotion of G 2 /M phase transition, inhibition of G 2 DNA checkpoints, and induction of defects in mitotic spindle checkpoints 43 . Herein, by treating the MCF-7 cells with the IC 50 of compound 5a the percentage of the cells was significantly arrested at sub-G0/G1 at a rate of 48.5%, compared with that of the untreated control MCF-7 cells (8.6%). These results confirmed the apoptosis of the MCF-7 cells. www.nature.com/scientificreports/ Blocking of the RAS/PI3K/Akt/JNK signaling pathways in MCF-7 cells by compound 5a. The elucidation of intracellular signaling pathways and the identification of oncogenes have ushered in a new era of targeted therapy that has greatly improved the prognosis for many malignancies. New therapies targeting these proteins and their downstream mediators have established their capability to stabilize and shrink tumors 44 . RAS oncogenes are the most frequently mutated oncogenes in human cancer, and RAS-mutant malignancies account for a significant portion of all human diseases 45 . RAS proteins cycle between two conformational states, that is, the active form when they are bound to GTP and the inactive form when bound to GDP. Guanine nucleotide exchange factors (GEFs) are known to stimulate GTP-bound RAS formation, whereas GTPase-activating proteins (GAPs) promote GTP hydrolysis on RAS thereby returning them to the inactive state 46 . In numerous human malignancies, including breast carcinoma, active forms of RAS have been found to induce cellular transformation. Thus, one of the therapeutic approaches to treating breast cancer is the efficient control of oncogenic RAS 47 . In this study, western blot analysis revealed that treatment with the IC 50 of 5a significantly decreased the levels of p-RAS proteins as compared to the MCF-7 untreated cells. This suggested the antiproliferative nature of this novel compound (Fig. 9). www.nature.com/scientificreports/ Activated RAS enhances cellular proliferation by modifying a wide range of intracellular effector pathways that eventually converge to promote growth and suppress apoptotic signals in transformed cells. The PI3K-Akt pathway has been described as a dominant growth survival pathway in breast cancer. The elevation of PI3K-Akt signaling is a common mechanism that malignant cells require to proliferate and escape programmed cell death 48 . Hence, in the present study, we examined the effect of 5a on the RAS/PI3K/Akt signaling pathway by assessing PI3K, Akt, and p21 gene expression as well as p53 protein expression in MCF-7 cells.
RAS protein interacts with PI3K, thereby, resulting in the phosphorylation and activation of Akt 46 . PI3K phosphorylates inositol phospholipids and generates the secondary messenger, phosphatidylinositol-3,4,5-triphosphate (PIP3), which triggers the phosphorylation and activation of the downstream serine/threonine kinase Akt. In turn, Akt promotes cell survival by modulating key proteins with various biological functions 49 . In this study, the IC 50 of 5a treatment significantly reduced mRNA transcript level of PI3K and Akt compared with the MCF-7 untreated cells which strongly indicated the inhibitory effect of 5a on the RAS/PI3K/Akt pathway (Fig. 8).
Additionally, phosphorylated Akt is a well-established cell survival factor because it downregulates both p21 and p53 50 . In this work, the treatment with the IC 50 of 5a significantly upregulated p21 gene expression compared with the untreated cells (Fig. 8). Moreover, the immunoblot analysis confirmed a significant elevation of p53 protein expression in the 5a-treated cells compared with that in the MCF-7 untreated cells (Fig. 9). www.nature.com/scientificreports/ After the treatment with 5a, the upregulation of p21 gene expression and p53 protein expression confirmed the antineoplastic effect of 5a toward human breast cancer cells by suppressing the RAS/PI3K/Akt signaling pathway. Furthermore, RAS is known to activate the JNK/MAP kinase signaling cascade leading to the phosphorylation of c-Jun and augmentation of the activator protein-1 (AP-1) transcriptional activity. Hence, we investigated the effect of 5a on the cell survival pathway, that is, the RAS/JNK pathway. Our study showed a significant reduction in the p-JNK protein expression when compared with that in the MCF-7 untreated cells suggesting the inhibitory effect of 5a on the RAS/JNK pathway (Fig. 9). These results may have been obtained because JNK contributes to the RAS-induced estrogen-independent growth of breast cancer cells. Additionally, the JNK pathway is involved in the expression of matrix metalloproteinases (MMPs) that are associated with extracellular matrix degradation and tumor metastasis. Moreover, there is an association between the JNK/c-Jun activation and angiogenesis in invasive breast carcinoma 51 . Indeed, JNK inhibition can cause apoptosis of tumor cells 52 .
SAR (structure-anti cancer activity relationship). For novel synthesized compounds, the in vitro anti-cancer and molecular docking results showed the following structure activity relationship (Fig. 10): • The presence of a bioactive pyrazole ring increases the anticancer activities 53 .
• (-OH) and (-OCH 3 ) that was found at the aromatic ring improved the anticancer activities. www.nature.com/scientificreports/ • The presence of pyrimidine ring is considered an essential moiety for enhancing anticancer activity 54 .
• The high aromaticity of the prepared compounds enhanced the anticancer activities.

Conclusion
Pyrimidine-2-thione derivatives 2-6 were successfully synthesized via conventional and ultrasonic irradiation techniques. The ultrasonic technique was found to be more effective than conventional heating, as evidenced by its higher yields and short reaction times. compound 5a was selected among the others according to their in silico molecular free binding energy docking studies towards inhibiting the H-RAS target protein and obeyed Lipinski's rule of five. Additionally, the results of the in vitro studies emphasized that the newly synthesized compound 5a induced cell death via targeting the RAS/PI3K/Akt signaling pathway. Moreover, it upregulated p21 and p53 proteins in the human breast cancer cells. Finally, compound 5a exhibited antineoplastic activity via the inhibition of p-JNK protein in cancer cells. Therefore, 5a appears to be an attractive antitumor compound with future clinical applications for breast cancer treatment (Fig. 11). www.nature.com/scientificreports/     53 Shamim etal., 54 Shamim etal., 54 Shamim etal., 54 Figure 10. Rational design of newly synthesized pyrimidine-2-thione derivatives. www.nature.com/scientificreports/

Data availability
The datasets generated and/or analysed during the current study are available in: Macromolecule protein structure, can be deposited in the worldwide protein data bank repository, (https:// www. rcsb. org/ struc ture/ 5P21). mRNA sequences, can be deposited in the National Center for Biotechnology (NCBI), (NM_181524.